The present invention relates to a carotenoid comprising preparation. The preparation of the invention can be used in food, in cosmetics, in a variety of medicinal uses, etc. The invention also concerns a method for the production of the carotenoids.
Carotenoids are pigments produced by microorganisms, fungi and plants, and used by them as antioxidants and protectants against excessive radiation. The most widely used carotenoids in food, medical preparations or cosmetics are xcex2-carotene and lycopene. xcex2-Carotene and lycopene are sensitive to light and oxidation, a property which considerably limits their use and shortens shelf-life of products containing them (in: Carotenoids, Chemistry and Biology, Krinski, N. I., Matthews-Roth, M. M., Taylor, R. F., ads), Planum Press, New York, London, 1989). In addition, xcex2-carotene and lycopene have a distinctive orange color and this color has a serious limitation for a variety of cosmetic or food applications.
Phytoene (7, 8, 11, 12, 7xe2x80x2, 8xe2x80x2, 11xe2x80x2, 12xe2x80x2-octahydro-xcex3,xcex3-carotene) and, phytofluene (15Z, 7, 8, 11, 12, 7xe2x80x2, 8xe2x80x2-hexahydro-xcex3,xcex3-carotene), are carotenoids (C-40 isoprenoids chain) which are precursors in the biosynthetic pathway which leads to the production of xcex2-carotene, lycopene and other carotenoids (phytoene is the first carotenoid-specific precursor and phytofluene is produced therefrom in a subsequent desaturation step). Phytoene is completely colorless whereas phytofluene has a slight yellowish color. Japanese Patent Application No. 90-40520, disclosed that the introduction of a DNA sequence yielding expression of phytoene into certain transfected cancer cells, resulted in inhibition of their growth and in inhibition in the activation of Epstein Barr virus (EBV).
In accordance with the present invention it was shown that phytoene and phytofluene possess anti-oxidative properties and furthermore are capable of absorbing ultra violet (UV) light. In addition, although having these properties, phytoene and phytofluene were found to be much more stable to oxidation than, for example, xcex2-carotene. These findings led to the realization that phytoene and phytofluene in combination could be useful in the prevention of environmentally induced damage of various kinds.
Thus, in accordance with the invention there is provided a composition comprising, an amount of phytoene and an amount of phytofluene, effective in combination in the prevention of damage resulting from oxidation and from exposure to UV light.
In accordance with the invention, the term xe2x80x9cdamagexe2x80x9d is to be understood as any damage resulting from a variety of oxidative agents such as oxygen itself, hydroxyl radical, hydrogen peroxide, other free radicals, ozone etc., or from any kind of harmful UV irradiation, such as UVA and UVB irradiation. The damage would depend upon the target for which the preparation is used. Thus, if the preparation is used on skin, damage may be any skin damage such as burns, blisters, damage appearing after chronic exposure to sun, e.g. premature aging of the skin, etc. When the preparation is used as a food preservative, such damage may be in the form of a decrease in product stability, chemical modification resulting for example in rancidity, accelerated aging, etc.
In accordance with a preferred embodiment of the invention there is provided a topical skin composition for protecting the skin against environmental hazards, comprising phytoene and phytofluene in an effective amount such that, in combination, these carotenoids exert an oxidation-protecting and UV-protecting effect on the skin.
The term xe2x80x9cenvironmental hazardsxe2x80x9d relates to any environmental agent which can exert damage such as UV radiation or oxidative agents.
The term xe2x80x9ceffective amountxe2x80x9d should be understood to mean an amount of phytoene and an amount of phytofluene which, when administered in combination, achieves the desired protective effect.
The phytoene and phytofluene in the composition of the invention may each be either in their trans or in their cis forms.
The weight ratio between the phytoene and phytofluene in the composition of the invention can range between 200:1 to 1:200, respectively, typically between about 50:1 to 1:50, preferably from 10:1 to 1:10, 10:1 (phytoene:phytofluene) being a particular example. The above ratios of phytoene to phytofluene may be reached either by using an extract which contains both carotenoids in the desired ratio, by adding an additional amount of one of the carotenoids to an extract comprising both carotenoids so that the desired ratio is obtained or by mixing the two separate carotenoids (each obtained by any of the methods mentioned or described above and below) to reach the desired ratio between them.
One of tie novel features of the inventive composition is that while possessing the above noted properties, the combination of phytoene and phytofluene is essentially devoid of any color (but for a slight yellowish hew, hardly visible, of the phytofluene). The composition of the invention will have less color than any other composition comprising a combination of carotenoids when compared at similar concentrations of carotenoids. The fact that the composition is essentially colorless ensures that these carotenoids will not have any effect on the aesthetic properties of the preparation comprising them. In addition, the lack of absorbance of light in the visible range (which is a manifestation of the fact that they are essentially colorless) renders them stable to degradation under visible light.
The preparation may, in accordance with the above noted preferred embodiment, be used as a topical cosmetic or pharmaceutical preparation in order to protect the skin from environmental hazards such as those described above including UV (UVA and/or UVB) irradiation or damages which can be effected by a variety of oxidative agents. A topical composition of the invention may be in the form of a gel, an oil-in-water or water-in-oil emulsion, a salve or ointment, etc.
In accordance with another embodiment, the composition may be used as an additive in food preparations, e.g. serving as a preservative to protect against oxidation of the various food ingredients, e.g. of oils or fats.
At times, the composition of the invention may comprise additional components which do not substantively change the basic characteristics of the composition. One example of such a component is zetacarotene (7,8,7xe2x80x2,8xe2x80x2-tetrahydro-xcex3,xcex3-carotene).
The composition of the invention may obviously, depending on its use, comprise also other ingredients, cosmetical or pharmaceutical acceptable carriers, preservatives, other antioxidants, various pharmaceutically or cosmetically active ingredients such as a topically acting drugs, etc.
In accordance with one preferred embodiment, the composition comprises also a hydrophobic carrier, which may be selected from oils typically used in the cosmetic, pharmaceutical or food industry, such as vegetable, mineral or synthetic oils.
The phytoene and phytofluene may be obtained from a variety of sources. Typically, they may be obtained from organisms that produce carotenoids, such as a variety of plants, various algae, and particularly micro algae Dunaliella sp, being a specific example. Very low amounts of phytoene and phytofluene have been produced by xcex2-carotenoid producing organisms. To obtain even such low amounts of the carotenoids, the organisms were grown under dim light conditions. In accordance with the invention, a method is provided which enables to yield substantive amounts of at least one of phytoene or phytofluene from such xcex2-carotenoid producing organisms. The term xe2x80x9csubstantive amountsxe2x80x9d relates to an carotenoid producing organisms. The term xe2x80x9csubstantive amountsxe2x80x9d relates to an amount of phytoene or an amount of phytofluene ranging from about 0.1 mg/l culture to about 30 mg/l culture typically between about 1 mg/l to about 20 mg/l. When the carotenoid producing organism is the algae Dunaliella sp., the typical amount of phytoene and phytofluene which may be produced is between about 1 mg/l to about 15 mg/l. In accordance with the method of the invention, the carotenoid producing organism, typically algae, may be grown under various kinds of conditions.
In accordance with one embodiment of this aspect of the invention, the carotenoid producing organisms are grown outside, for example, in the case of Dunaliella sp. in outside pools in a growth medium comprising sea water and recycled salt water at a temperature of between about 10xc2x0 C. to about 40xc2x0 C. The organisms may be grown under sunlight at an extent of between 80% shade to fill sunlight By a preferred embodiment, the carotenoid producing organisms are grown under bright light ranging from about 40% shade to full sunlight.
By an additional embodiment, the carotenoid producing organisms are grown in a growth culture, typically, in a fermenter and typically at room temperature in which case the growth medium will typically comprise various salts and minerals in different combinations (see, for example, Example 1.1 below). In accordance with this embodiment, the organisms are grown under broad spectrum light (light spanning over a substantive portion of visible light) of above about 10 W/m2 and which may range from about 10 W/m2 to about 200 W/m2.
In accordance with the method of the invention, substantive amounts of phytoene and/or phytofluene are typically produced following several days of growth of the organism (e.g. about 4 to 6 days).
The phytoene and phytofluene are obtained under conditions which favor the accumulation of phytoene and phytofluene in cells of the organisms, typically by growing a xcex2-carotene-producing organism in the presence of carotenoid-biosynthesis inhibitors, which are inhibitors that can act in subsequent reaction steps in the biochemical pathway of the production of carotenoids. One example of inafter: xe2x80x9cthe 4-chloro inhibitor xe2x80x9d). When the source of phytoene and phytofluene is the micro algae Dunaliella, the 4-chloro inhibitor is typically included in the growth medium in a concentration of about 0.1 xcexcM (Ben-Amotz et al, Plant Physiol. 86:1286, 1988). Other inhibitors which may be used are, for example, J334, (Ben-Amotz et al. Supra) Sandoz H 6706 (Kxc3xcmmel, H. W. et al., Z. Naturforsch 30c, 333, 1975), di-phenyl-amine (DPA) (Foppen F. H., Ann 1st. Super. Sanita, 439, 1969) and nicotine (Shaish et al., Plant Cell Physiolo 31:689, (1990)).
In accordance with one embodiment of this aspect of the invention there is thus provided a method for preparing a composition comprising at least one of phytoene or phytofluene, comprising the steps of:
(i) incubating a carotenoid producing organism in a growth culture under broad spectrum light having an intensity of above about 10 W/m2 in the presence of one or more carotenoid synthesis inhibitors;
(ii) growing said carotenoid producing organisns until the level of at least one of phytoene or phytofluene is between about 1 mg/l culture to about 50 mg/l culture; and
(iii) separating said organisms from the culture.
In accordance with an additional embodiment of this aspect of the invention there is provided a method for preparing a composition comprising at least one of phytoene or phytofluene, comprising the steps of:
(i) Incubating a carotenoid producing organism in a growth culture under sunlight at an extent between about 80% shading to full sunlight in the presence of one or more carotenoid synthesis inhibitor;
(ii) Growing said carotenoid producing organisms until the level of at least one of phytoene or phytofluene is between about 1 mg/l culture to about 50 mg/l culture; and
(iii) separating said organisms from the culture.
In accordance with this aspect of the invention, the phytoene and phytofluene obtained in the grown organisms may be used without being separate,
In accordance with this aspect of the invention, the phytoene and phytofluene obtained in the grown organisms may be used without being separated from the dry matter of the producing organism. However, in accordance with a referred embodiment, following separation of the carotenoid producing organism from the culture, a preparation is further prepared from the organisms comprising phytoene, phytofluene or both. Typically, the preparation is an extract which may be prepared from the carotenoid producing organisms by any of the extraction methods known in the art such as, for example, by ethanol:hexane extraction.
In accordance with the method of the invention, typically, both phytoene and phytofluene are obtained from the carotenoid producing organism and the ratio of phytoene to phytofluene may vary according to the conditions under which the organism was grown and, when a preparation is prepared, the type of methods used for obtaining the preparation. At times, however, under certain conditions, growing the carotenoid producing organism in accordance with the method of the invention may result in obtaining mostly one or only one of the two carotenoids.
In accordance with the invention it was also found that the carotenoids ratio may further be enhanced in favor of phytoene and phytofluene by the addition of active charcoal during carotenoid extraction. Charcoal may be added when an inhibitor is used as well as when the carotenoid producing organism is grown without the inhibitor. The charcoal is later filtered out from the culture or preparation using any of the known centrifugation or filtration methods.
In addition to the above, in accordance with the invention the carotenoids in the composition of the invention may also be synthesized by any of the known chemical or biochemical methods or recombinant methods. Chemically, phytoene can be synthesized, for example, from two geranylgeranyl pyrophosphates (C-20), in a reaction which may be mediated by phytoene synthase. The geranylgeranyl pyrophosphate can be obtained directly, by the conversion of mevalonic acid or by the condensation of pyrovate and glyceraldehyde-3-phosphate. Phytofluene can be synthesized by desaturation of phytoene, a reaction which may be mediated by phytoene desaturase. Recombinant methods include, for example, the mutagenesis of enzymes which are active downstream to phytofluene. Such synthesized phytoenes and phytofluenes will have activities which are substantively similar to the activities of the phytoene and phytofluene obtained from organisms that produce carotenoids as explained above.
The stability of the carotenoids in the composition of the invention can be tested by irradiating the carotenoid composition by light and/or exposing the composition to oxygenating agents. The effect of such treatments on the carotenoids in the compositions of the invention, is less than the effect of such treatments on other carotenoids such as, for example, xcex2-carotene. In prior art compositions containing an effective amount of carotenoids, such treatment typically result in degradation of the carotenoids, manifested for example by change in light absorbance, i.e. color. The anti-oxidative effect of the composition of the invention can be determined, for example, by determining the anti-oxidation protection effect on DNA (Salles et al., Anal. Biochem., 232:37, 1995).
The invention will now be illustrated firer in the following description of the non-limiting specific embodiments.